Due to the advances of recombinant DNA technology, proteins that were normally produced in small quantities by organisms in nature and or were difficult to purify from such organisms can now be produced in large amounts. Typically, the gene which causes the proteins to be made in nature is inserted into the DNA of bacterial, yeast or mammalian cells; the cells cultured; and the protein purified from the cells after sufficient growth.
However, the nature of the cells used to produce the protein of interest (subject protein) can limit their application or require expensive equipment and reagents. For example, proteins expressed by higher organisms often undergo modifications after they are initially expressed by the cells. Such modifications are referred to as glycosylation. Glycosylation of proteins produced in the cells of mammals and other higher organisms in nature is often necessary for such proteins to elicit biological activity.
However, bacterial cells are incapable of glycosylating proteins. Consequently, they often cannot be used to make proteins which are intended to elicit biological activity in mammals, fish, and insects.
Although mammalian and insect cell systems can be used to manufacture glycosylated proteins, expensive and complex media are required and the bioreactors, in which the cells are grown, must be run for extended periods creating a risk of contamination of the cell culture.
Because insect larvae can be grown quickly and inexpensively, there have been attempts to genetically engineer them to express a subject protein instead of using cells to produce a subject protein. The fact that yields can be obtained from insect larvae which cannot be obtained from bacterial cells also makes them an appealing alternative to cell based protein manufacturing.
An additional reason for utilizing insect larvae is that a system is available which can readily be used to genetically engineer them. Baculoviruses characteristically have a circular double-stranded DNA genome which is contained in a rod-shaped enveloped virion. The DNA can be manipulated to incorporate a gene which encodes a subject protein. Like all viruses, the DNA of the baculovirus will cause the cells of its host to produce the proteins encoded in its DNA. Consequently, if the DNA of a baculovirus is manipulated to incorporate a gene which codes for a subject protein and that baculovirus allowed to infect an insect larvae, the cells of that larvae will produce the subject protein.
Attie et al., U.S. Pat. No. 5,472,858 disclosed this approach with the tobacco hornworm. After the hornworm is infected with a recombinant baculovirus, it begins secreting the recombinant protein into its hemolymph. The hemolymph can then be withdrawn using a syringe throughout the larvae's growth.
However, there is a drawback to this specific method. Although the tobacco hornworm larvae is ideal for the physical manipulation because of its large size, a great deal of manual labor is required to extract the recombinant protein if large numbers are to be cultivated.
Furthermore, in this method, the individual larvae are injected with baculovirus to initiate infection, which can also be labor intensive.
Besides this problem, many larvae produce cellular and digestive proteases when they overexpress genes, as in the case of recombinant engineering with baculoviruses. It is the function of proteases to break down various proteins in the larvae. As a result of the expression of these proteases, the subject protein could be consumed while in the larvae.
Additionally, in many insect species, recombinant proteins cannot be recovered from the larvae after a certain point because the baculovirus eventually kills its insect host. In the larvae of Trichoplusia ni, more commonly referred to as the cabbage looper, the larvae die after five to seven days after infection with the baculovirus and melanization occurs. During melanization, many of the proteins condense into a dark, gluey mass from which recovery of the recombinant protein is impossible.
Thus, the harvest of the larvae currently has to be synchronized with the viral infection cycle and their life cycles to obtain an optimal yield. However, all the larvae in a population will not have the same rate of growth and viral infection since characteristics, such as individual growth rates, can vary widely among a population. Consequently, although all of the larvae in a production population may have been infected with baculovirus at the same time, some will not have attained their optimal level of protein expression when the entire population is harvested simultaneously.
Sacrifice and assay of individuals within the population of a production run might be a method to determine when most of the larvae have obtained the optimal expression of the subject protein. However, due to random variations of individual characteristics in a population, the individuals selected may not be representative of the entire population.
Large samplings may result in a more accurate profile of expression with the population, but this is time consuming and labor intensive and will leave less larvae for harvest.
Moreover, this "batch" run method, where larvae are synchronously infected and harvested, requires an entirely new run to be started to make up for any deficiencies in yield that become apparent from an existing production run. That is, if a "batch" run is showing signs of a poor yield, adding more larvae would be impractical since their infection cycles will not be synchronized with those of the already infected larvae. The result being that the optimal level of protein expression in the new larvae will not be obtained when the entire population is harvested.
The preferred alternative to the "batch" run approach is a "continuous" approach where larvae can be continually added to and harvested from a production population regardless of the stage of infection or growth of other larvae in the population.
From the above, there is a need for an insect larval protein manufacturing system that allows easy selection of individual larvae for harvest at their optimal point of protein expression with minimal labor that does not require synchronous infection and harvest.